Cellular proteins are now appreciated as critically involved in all steps of the human immunodeficiency virus type 1 (HIV-1) life cycle, and disrupting host functions essential for virus replication may provide novel antiviral approaches. To identify new molecules in human cells critical for blocking HIV-1 replication we established a genetic screen of mammalian complementary DNAv(cDNA) libraries for genes that prevent infection by a genetically marked retrovirus. A cDNA library from human cells (HeLa) stimulated by interferon (known to activate many antiviral proteins) was generated, introduced into a human virus-susceptible cell line, and a virus resistant population was selected for survival after multiple rounds of infection with an HIV vector expressing a toxic gene. Two active cDNAs were identified, dubbed H1 and H2: H1 expresses the N- terminal portion (86a.a.) of heterogeneous nuclear ribonuclear protein U (hnRNP U) and H2 the N-terminal portion (93 a.a.) of the eukaryotic initiation factor 3 subunit p47 (elF3p47 or elF3f). hnRNP U is involved in pre RNA processing, mRNA transport to the cytoplasm, intracellular localization, and turnover of mRNAs. elF3f belongs to the elF3 complex that plays a role in translation by associating elF2-GTP-Met-tRNA and promoting RNA binding. elF3f is also related to a mouse protein called Mov34;the human homologue of Mov34 is the S12 subunit of the 26S proteasome. Both cDNAs were characterized as necessary and sufficient to confer resistance to HIV infection. However, H1 expressing cells seems to induce retention of the viral mRNA in the nucleus, while H2 expressing cells induce a drastic reduction in the amount of the nuclear viral mRNA by specifically reducing cleavage activity. In both cases levels of the viral messages in the cytoplasm are dramatically reduced. Both gene fragments specifically target the 3'long terminal repeat 3'LTR) in the viral mRNA. These results suggest that HIV-1 requires machinery for the nuclear export/cleavage of the mRNA message that can be specifically inhibited by the identified cDNAs. We propose to continue and expand these investigations, as well as further identify more interfering factors by screening cDNA, peptide or RNA interfering (RNAi) libraries. The discovery of cellular factors involved in retroviral replication and an increased knowledge of their mode of action may lead to important antiviral approaches in clinical settings.